This is a resubmission seeking renewal of a 25-year program encompassing 4 sub-projects. The present application sharpens the focus of the laboratory on the pulmonary circulation, by a multi-faceted study of the mechanisms of hypoxic pulmonary circulation, by a multi-faceted study of the mechanisms of hypoxic pulmonary hypertension (PH). The work is collectively founded on the hypothesis that PH smooth muscle cell (SMC) function. Studies of functional contributions explore the influence of hypoxia on the ratio of pulmonary vasoconstrictors/dilators and of altered ion channel function . Work exploring structural aspects tests the idea that there exist specific subpopulations of hypoxia-responsive SMCs responsible for a preponderance of remodeling of lung vessels in hypoxia and that hypoxic proliferative response is dependent on specific isoforms of protein kinase C. The projects share experimental models of hypoxic pulmonary hypertension in rats and cows, as well as 3 core laboratories. This project will test 3 specific aims: 1) If hypoxic pulmonary hypertension is associated with increased endothelial NOS but not ET-1 protein, whereas the opposite is true in genetic pulmonary hypertension; 2) If the mechanisms of action of nitric oxide synthase (eNOS) and ET-1 gene expression are hypoxia and hemodynamic stress, respectively in these models. This project will investigate 4 specific aims: 1) What changes in PA SMC ion channels develops as a result of hypoxic pulmonary hypertension; 2) How does Ca2+ enter the hypertensive PA SMC; 3) How do NO and cGMP regulate ion channels in the hypertensive PA SMC; and 4) Are changes in ion channel expression and regulation a direct result of chronic hypoxia. This project will test 3 hypothesis: 1) Gi coupled receptors activate the MAPK signaling pathway in selected populations of PA SMCs; 2) Hypoxia selectively stimulates in hypoxia-responsive PA SMCs through MAPK-dependent mechanisms; and 3) Connective Tissue Growth Factor (CTGF), is expressed selectively in subpopulations of PA SMCs during hypoxia and contributes to the fibroproliferative response. This project will test 2 specific aims: 1) Are PKC-alpha expression and activation critical determinants of PA SMC proliferation to hypoxia in vivo, and 2) Are PKC-alpha expression and activation critical determinants of PA SMC proliferation to hypoxia in vitro. The 4 projects are highly interactive both conceptually, as well as in the performance and communication of experimental results.